Expression of Wwox, a likely breast cancer tumor suppressor gene, is lost or reduced in the majority of invasive breast cancers and DCIS, as well as in some normal appearing glands in 1/3 of breast cancers, suggesting that Wwox loss is an early, perhaps predisposing event in breast cancer. Wwox protein, through its first WW domain, interacts with transcription factors p73, Ap2alpha and gamma, ErbB4, and affects their transcriptional activity. Knockdown of Wwox expression in MCF7 breast cancer cells abrogates the in vitro tamoxifen response and, conversely, tamoxifen resistant MCF7 clones show reduced Wwox expression. Immediate objectives of this proposal are to: define the mechanism of tamoxifen resistance in cancer-derived cell lines, confirm the pathway in cancer tissues, and thus identify markers that may determine which cancers will be sensitive to tamoxifen therapy; map the extent of WWOX promoter methylation throughout cancerous mastectomy specimens, as a marker for breast cancer field cancerization. The proposed research is based on the interconnected hypotheses that Wwox expression level is a critical determinant of the response of breast cancers to tamoxifen in ERalpha positive tumors and that Wwox loss is an early determinant of breast cancer. The long-term objective is to establish Wwox as a central mediator of hormone response and therapy of breast cancer, a mediator that can be reexpressed through epigenetic therapy, to activate or maintain tamoxifen sensitivity. The aims are to: 1. establish the relationship of Wwox expression to tamoxifen response by: a) up- and down-modulation of Wwox in breast cancer cells and examination of effect on tamoxifen response; b) use of Wwox WW domain mutants that abolish Wwox binding to interacting transcription factors in similar experiments; c) restore ERalpha expression in Wwox positive and negative cells and examine effect of tamoxifen; d) examine expression levels of PKA and PKA-Rlalpha relative to Wwox expression level; e) determine effect of Wwox reactivation, by demethylating agents, on tamoxifen response. This aim will confirm the critical role of Wwox in modulating the tamoxifen response and show that tamoxifen sensitivity can be reestablished in Wwox negative breast cancers by epigenetic therapies. 2. Determine mechanism of Wwox mediation of tamoxifen signals by a) examining expression and subcellular localization of known Wwox binding partners, in tamoxifen sensitive and refractory breast cancers; b) seeking a mechanistic connection between loss of Wwox expression and reduced PKA-Rlalpha expression; c) identifying novel interactors by coimmunoprecipitation and examining role in tamoxifen response, if known Wwox interactors are not implicated in resistance. This aim will identify the critical Wwox binding proteins that mediate tamoxifen response. 3. Establish the in vivo relevance of Wwox and its binding partners in breast cancer by determining expression levels and subcellular localization of ERalpha, PR, Wwox, ErbB2, Ap2alpha, Ap2gamma, p73, ErbB4, PKA and PKA-Rlalpha in tamoxifen sensitive and resistant breast cancers: a) by selection of panels of tamoxifen sensitive and refractory breast cancers and reassessment of ER, PR and ErbB2 status; b) immunohistochemical evaluation of expression and subcellular localization of Wwox interactor proteins. This aim will confirm, in vivo in breast cancers, the role of Wwox in mediating expression of such proteins as ErbB2, and expression and subcellular localization Wwox interactors whose function controls tamoxifen response. 4. Map the extent of WWOX promoter methylation throughout surgically removed cancerous breast tissues. This aim will determine if WWOX silencing is a marker of a field cancerization effect. [unreadable] [unreadable] [unreadable] [unreadable]